Close-range interaction with the bone marrow microenvironment plays major role in the regulation of hematopoietic stem cell (HSC) activity. Microenvironmental support of hematopoiesis is mediated by non-hematopoietic or stromal elements, which also support HSC proliferation in long term bone marrow culture (LTBMC). Stromal cultures appear heterogeneous, producing a complex extracellular matrix of collagens, glycoproteins and proteoglycans (PG's). There is evidence that PG's may contribute to mediation of the hematopoietic microenvironment and it is therefore proposed to characterize hematopoietic stromal PG's. Cellular heterogeneity limits the feasibility of stromal PG characterization in intact LTBMC. Two well-documented continuous stromal cell lines (1-2) will therefore be used in addition to primary stromal cultures. Stromal cells will be labeled with 35S-sulfate, 3H-glucosamine and 3H-serine. Heterogeneity of PG's will be investigated by ion exchange chromatography, gel filtration chromatography and isopyknic cesium chloride centrifugation. Studies will be carried out on each PG identified to determine its glycosaminoglycan (GAG) side chain species, hydrodynamic size and sulfation. Oligosaccharides will also be characterized. Core protein hydrodynamic size will be determined and proteolytic digestion studies performed with various enzymes to determine GAG and oligosaccharide distribution on the protein core. The subcellular localization of PG's will be determined and the kinetics of PG synthesis, cellular transport, secretion and breakdown will be investigated in pulse-chase experiments. Glucocorticoids and xylosides have profound effects on hematopoiesis in LTBMC. The effects of these agents on PG synthesis in stromal cell lines and in primary stromal cultures will therefore be investigated.